Effect Of FtsZ Interacting Proteins On Cell Division And Nucleoid Compaction In Escherichia Coli

The eukaryotic tubulin homologue FtsZ polymerizes into a Z ring,which serves as a scaffold for the recruitment of downstream division proteins leading to cell division.In this study,we found that GFP-FtsZ immunoprecipitation experiments show that FtsZ interacts with the cell division protein AmiA,Chaperonin GroEL,S-adenosylmethionine synthase MetK,and Chromosome initiation inhibitor ArgP in vitro.We have verified that FtsZ is indeed compatible with AmiA,GroEL,MetK,and ArgP in vivo through bacterial two-hybrid experiments.The predicted structure of each protein which interacts with FtsZ is determined.Upon mutations by site-directed mutagenesis in the a-helix,b-sheet and linker region on the plasmid,the colonies grown by the transformants carrying p KN25-fts Z and p UT18-ami A^N97K,p UT18-gro EL^K28N,p UT18-metK^K47N,and p UT18-arg P^N44K are white by using bacterial two hybrid system,which indicates that FtsZ loses the ability to interact with these proteins.The effects of these proteins on cell size and nucleoid morphology and distribution were examined by deleting and overexpressing them.We found that whether it is LB or ABTGcasa medium,the deletion of ami A gene will lead to an increase in cell length,indicating that AmiA is essential for cell division.After the pami A plasmid is transferred into the mutant,the increase in cell length was not reversed.The presence of excess AmiA cause a slight increase in the length of wild type cells.The deficiency or overexpression of AmiA protein greatly exacerbate the nucleoid compaction in the cells.The mutations made by site directed mutagenesis in the AmiA protein also cause an increased cell size and unorganized nucleoid compaction.These results indicate that AmiA affects cell size and nucleoid compaction.In the absence of metK,cells increase slightly in LB and ABTGcasa medium.After the pmetK plasmid is transferred into the mutant,the cell size in the?metK cells is reversed.However,overexpression of MetK^K47N does not reverse the cell phenotype.Interestingly,MetK^K47N does not interact with FtsZ anymore,indicating that MetK availability affects cell size and nucleoid compaction through interacting with FtsZ.The nucleoid compaction depends upon the availability of MetK.In the absence of ArgP,cell length increases in LB and ABTGcasa medium.Upon growing excess ArgP in BW25113 cells,cell size is not affected.The mutation in ArgP^N44K made by site directed mutagenesis does not reverse the cell size of?arg P to wild-type cells and nucleoid compaction,at the same time,not interact with FtsZ.These results indicate that correct cell division is dependent on ArgP’s interaction with FtsZ.The absence or excess ArgP leads to unorganized nucleoid compaction.Deletion or overexpression of GroEL does not affect cell size but affect the nucleoid compaction,so is the mutation GroEL^K28N.In summary,AmiA,MetK,and ArgP proteins interact with FtsZ and affect cell size and normal nucleoid compaction.AmiA^N97K、MetK^K47N、ArgP^N44Kand GroEL^K28Nmutants no longer interact with FtsZ and no longer alleviate the cell division problem of?metK and?arg P mutants.These results indicate that MetK and ArgP affect cell division through interaction with FtsZ,and the detailed mechanism remains further study.